Introduction
Herpes simplex virus 1 (HSV-1) has a double-stranded DNA genome of about 152 kbp, from which more than 84 ORFs are translated. Since Post and Roizman first characterized recombinant viruses in which a specific HSV-1 gene was mutated by the reverse genetics system [
1], this gene's roles in the viral life cycle have been extensively investigated. By now, there remain only a handful of HSV-1 genes whose roles have not been investigated using a recombinant virus with a mutated gene. The UL7 gene, the subject of this study, is one such viral gene. The UL7 amino acid sequence is conserved in all
Herpesviridae subfamilies [
2], suggesting that UL7 homologues may play conserved roles in the herpes virus life cycle. The viral gene is on the left side of the HSV-1 unique long (U
L) region and surrounded by two essential viral genes (UL6 and UL8) for virus replication in cell cultures [
3]. The UL7 gene partially overlaps with the UL6 gene, and these transcripts are coterminal at their 3' ends. Information on the function(s) of the HSV UL7 gene product in the viral life cycle is limited. The only reported experimental evidence with regard to HSV UL7 is that its gene products are present in integumentary layers of mature virions, and that the viral protein is localized predominantly in the juxtanuclear cytoplasmic domains of infected cells, although it is also detected transiently in the nucleus [
4]. On the other hand, mutant viruses in which the UL7 homologous genes of other alphaherpesviruses pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1) have been constructed and characterized [
5,
6]. The mutant viruses revealed that the UL7 homologous genes are dispensable for viral replications of PRV and BHV-1, although the mutant viruses exhibit impaired capacity to replicate in cell cultures. These results indicate that the UL7 homologous genes of PRV and BHV-1 are involved in viral replication in cell cultures. However, the mechanisms underlying the actions of the gene products in viral replication are unclear. In the present study, we succeeded in generating a UL7 null mutant virus and characterizing it in cell cultures. Furthermore, as a first step to elucidating the mechanism by which UL7 functions in viral replication, we attempted to identify cellular proteins that interact with UL7.
Discussion
The essentiality of HSV-1 UL7 in viral replication in cell cultures has been controversial (Roizman & Knipe, 2001), and no experimental evidence supporting the assumptions of essentiality has been reported. In the present study, we have constructed a null mutant virus of HSV-1 UL7, called MT102, and presented evidence that MT102 is able to replicate in Vero cells, indicating that the HSV-1 UL7 gene is dispensable in HSV-1 replication in cell culture. Interestingly, both the plaque-forming ability and the viral growth of MT102 in cell culture were greatly impaired compared to those of the wild-type virus. This impairment of MT102's growth properties is due solely to the deletion of the UL7 gene, for two reasons: first, UL7 gene deletion did not affect the expression of neighboring genes UL6 and UL8, both of which are essential for viral replication in cell culture; and second, the repair of the UL7 gene deletion (MT103) restored the wild-type growth properties. These phenotypes of the UL7 null mutant virus of HSV-1 are consistent with those of PRV and BHV-1. Taken together, these observations indicate that UL7 is significantly involved in viral replication in cell culture.
In a previous report, electron microscopic analyses of the PRV UL7 null mutant virus demonstrated that the absence of PRV UL7 did not affect the intranuclear steps of virion formation, including capsid assembly, encapsidation of viral DNA, nuclear egress of capsids, and secondary envelopment in cytoplasmic membrane vesicles, but did affect the release of finally enveloped virions from cells [
5]. Consistently, the release defects of viruses have been observed with UL7 deletion mutant viruses of HSV-1 (this study) and BHV-1 [
6]. These results suggest that one of the conserved roles of UL7 homologues in viral replication is to regulate virion release from infected cells. On the other hand, virus titers in cells infected with MT102 were also impaired, as observed with the UL7 null mutant viruses of BHV-1 [
6] and PRV [
5], implying that each UL7 protein functions in at least one step of viral replication other than viral release. Thus, UL7 homologues seem to play multiple roles in viral replication. However, the mechanism or mechanisms by which the UL7 gene product acts in infected cells remain unknown.
As a first step to elucidate such mechanisms, we attempted to identify cellular protein interacting with HSV-1 UL7 by using the MS-based proteomics technology combined with a tandem affinity purification tag, called MEF [
9], and we identified ANT2 as a UL7-interacting partner. ANT is located in the inner mitochondrial membrane as a member of the permeability transposition pore (PT) complex, which comprises ANT, voltage-dependent anion channel (VDAC), hexokinase, and cyclophilin D, and regulates its functions so that they interact with each other [
3,
16]. ANT is a bifunctional protein that, in physiological conditions, exchanges ATP and ADP on the inner mitochondrial membrane, whereas in apoptotic conditions it can form a nonspecific pore [
3,
17]. Recently, ANT was reported to be a component of the mitochondrial permeability-transition pore (mtPTP); on the other hand, it is also essential for maintaining the cell metabolism exchange of cytosolic ADP for mitochondrial ATP [
16]. In the present study, we demonstrated that ANT2 from COS-7 cells transfected with the ANT expression vector and infected with wild-type HSV-1 was co-precipitated with UL7. Furthermore, UL7 is detected in both the mitochondrial and cytosolic fractions in infected cells in cell fractionation experiments, which reinforced the interaction between UL7 and the mitochondrial protein. Together, these series of observations indicate that UL7 interacts with ANT2 in HSV-1-infected cells.
The biological significance of the interaction between UL7 and ANT2 is uncertain. Four ANT proteins exist in human (ANT1~4) as the mitochondrial carrier family, and they are expressed in tissue- and development-specific manners [
18‐
20]. ANT2 is up-regulated in proliferative cells, including several cancer cell lines, and induces apoptosis by interacting with many kinds of materials [
21], including viral protein (Vpr of HIV-1 and pBI-F2 of influenza virus) [
22‐
24], although ANT2 was not an essential member. In addition, ANT2 repression results in the growth arrest of human cells; that is to say, only ANT2 negatively regulates apoptosis, and thus may be oncoprotein, despite the close similarity among the four ANT genes. ANT2 has therefore recently become a useful target for cancer therapy based on molecular targeting [
25]. These reports suggest the special involvement of ANT2 in conditions of stress, not only in cancer cells but also in viral infection. In addition, some mitochondrial changes in HSV-infected cells have been reported [
26,
27]. Spherical morphological change of mitochondria was observed using intensified fluorescence digital imaging at an early point in infection [
28]. A confocal microscopic study also reported clustering of mitochondria in HSV-2 infected cells [
29]. Oxidative stress of mitochondria and Ca+ release were observed by NF-kB activation induced by HSV infection [
30]. Other studies using HSV mutants, revealed the release of cytochrome C, which is known to be a stress-responsive mitochondrial protein, into the cytoplasm [
31,
32]; it thus confirms the influence of HSV infection on mitochondrial condition. From these facts, it is undeniable that UL7 may be involved in the control of mitochondrial functions and/or conditions through ANT2, because ANT2 is an important member of the mitochondrial inner membrane proteins that modulate mitochondrial life. It is also interesting that another ANT family member, ANT4 (SLC25A3), recently identified but with an unknown function [
19], also interacts with UL7 in human cells. Finally, UL7 may modulate the functions of ANT2 or some other ANT members and rescue HSV infected cells so that they can survive the virus on their own.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MT conceived this study, designed and performed the experiments and drafted the manuscript and writing. TS participated in the design of this study. YK participated in the design, coordination of this study. All authors read and approved the final manuscript.